吗啡耐受新说--受体寡聚体形成调节受体内吞与吗啡耐受

吗啡引起的耐受限制了其在慢性痛治疗方面的应用。阿片类物质的镇痛和依赖作用是由μ阿片受体所介导的。目前普     

Toll样受体4在吗啡应用和耐受中的作用

Toll样受体4(Toll-like receptor 4) 是主要表达于小胶质细胞表面开启哺乳动物先天免疫反应的重要受体.近年研究表明,TLR4参与疼痛及炎症的形成.TLR4 能够被吗啡激活,其结果导致小胶质细胞活化,细胞因子合成和释放增加,从而提高疼痛感受细胞的兴奋性,减小或抵消吗啡的镇痛作用,即形成吗啡耐受.抑制TLR4可以增加吗啡的镇痛作用,减缓吗啡耐受的形成.TLR4与经典阿片受体之间存在立体选择特异性差异,(-)和(+)吗啡均能使之激活.吗啡-TLR4-胶质细胞作用链的研究为治疗吗啡耐受产生提供新的路径.     

NMDA受体拮抗剂对阿片类药物耐受和依赖的阻断作用

本文综述阻断ＮＭＤＡ受体离子通道复合药物对阿惩耐受和成瘾发生的影响。行为药理学研究显示,非竞争性ＮＭＤＡ受体拮抗剂、竞争性ＮＭＤＡ受体拮抗剂和甘氨酸受占拮抗剂能抑制阿片耐受和戒断反应,其药理学特性明显不同于其他类型抗阿片耐受和成瘾的药物,阐述了ＮＭＤＡ受体拮抗剂治疗阿片类芗耐受和领事的系列化机制。并指出ＮＭＤＡ受体拮抗剂具有神经毒性。     

蛋白激酶C分子的成熟与活化

蛋白激酶C(Protein kinase C,PKC)是细胞内一类重要的Ser/Thr激酶,调控多种生命活动的信号转导过程,目前已发现了至少11种亚型,其结构有一定的保守性而又有所差别,导致其功能和调控的多样性。新合成的PKC一般需要经历活化茎环(Activation-loop,A-loop)、转角模体(Turn motif,TM)以及疏水模体(hydrophobic motif,HM)的程序性磷酸化过程才能成熟,获得进一步活化的功能。本文综述了近年来PKC的程序性磷酸化成熟以及活化的研究进展情况。     

蛋白激酶C分子的成熟与活化

蛋白激酶C（Proteinkinase C,PKc）是细胞内一类重要的Ser／Thr激酶,调控多种生命活动的信号转导过程,目前已发现了至少11种亚型,其结构有一定的保守性而又有所差别,导致其功能和调控的多样性。新合成的PKC一般需要经历活化茎环（Acti．vation-loop,A—loop）、转角模体（Tummotif,T1V1）以及疏水模体（hydrophobic motif,HM）的程序性磷酸化过程才能成熟,获得进一步活化的功能。本文综述了近年来PKC的程序性磷酸化成熟以及活化的研究进展情况。     

大麻素受体2在吗啡耐受中的作用

吗啡在疼痛治疗中广泛应用,但其长期使用可以导致耐受,这大大影响了其临床应用价值,吗啡耐受是临床亟待解决的问题。研究发现大麻素受体2(cannabinoid receptor 2,CB2受体)参与吗啡耐受的发生与发展。CB2受体选择性激活剂与吗啡联合使用,可以减弱吗啡诱导产生的痛觉过敏和异常疼痛,抑制吗啡耐受的发生与发展。激活CB2受体抑制吗啡耐受的机制尚未明确,本文将就CB2受体在吗啡耐受中作用的研究现状作一综述。     

吗啡调节免疫的分子机理

吗啡是阿片类的天然生物碱,常用的镇痛药物,作用于神经细胞表达的阿片受体,致使疼痛缓解和痛觉缺失。其次,吗啡对免疫系统功能有广泛的影响,对固有免疫和适应性免疫均起调节作用。吗啡抑制T淋巴细胞增殖及迁移、自然杀伤细胞活性,抑制免疫细胞产生MIP-1β、IFN-γ、IL-4、IL-10、IL-12等分子,并诱导CCR5、IL-1β、IL-6、TNF-α等。而对长期使用吗啡镇痛的肾细胞癌病人,同时使用褪黑素,不仅不影响吗啡的镇痛效果,还克服了其免疫抑制作用。吗啡调节免疫功能除了与阿片受体有关,还涉及许多的信号途径。现就吗啡对免疫系统调节作用的分子机理作概述。     

吗啡耐受大鼠心房心钠素的释放及其基因转录

本工作应用心钠素放射免疫测定和分子杂交技术首次发现,吗啡耐受大鼠血浆心钠素水平显著降低,心房内心钠素含量明显升高,同时心房内心钠素特异性mRNA水平也相应提高,提示在吗啡耐受时大鼠心房内心钠素的合成和贮存增加,释放减少。     

反复刺激下丘脑弓状核区引起耐受及其与吗啡镇痛的交叉耐受

电刺激下丘脑弓状核区具有明显的镇痛效应,这种镇痛效应随着反复刺激弓状核区次数的增加而迅速降低,以致消失。即产生了刺激弓状核区的耐受。并且,随着停止刺激后时间的延长,此耐受又逐渐消失,最后恢复至正常水平。同时,刺激弓状核区镇痛和吗啡镇痛存在双向交叉耐受现象。上述结果提示:刺激弓状核区镇痛和吗啡镇痛在作用机理上可能有相似之处,刺激弓状核区镇痛可能通过释放内源性阿片样物质(β-内啡肽)而起作用。     

蛋白激酶C与细胞周期

近年的研究表明,PKC涉及到细胞的周期调节。在酵母细胞和哺乳动物细胞均发现PKC参与细胞周期调控,从而提示PKC可能在进化上是一种保守的细胞周期调节子。一般认为PKC在两个点上对细胞周期起作用,即G1期和G2期到M期的过渡期（G2／M）。在G1期,PKC分别在早G1期和晚G1期作用有所不同,主要作用表现在使细胞停留在G1期的中末阶段,这一过程,主要涉及到抑制肿瘤抑制因子－成视网膜细胞瘤（Rb)蛋白的磷酸化。PKC的主要作用是降低周期素依赖激酶CDK2的活性、降低周期素E和A的表达和增加周期素依赖的周期抑制蛋白p21^WAF1和p27^KIP1的表达;在G2／M期,PKC对细胞周期的调节主要与Cdc2(CDK1)的活性抑制有关。     

Protein kinase C in rat brain myelin

It has been suggested that phosphorylation of myelin basic protein (MBP) in CNS is catalyzed by protein kinase C (PKC). In order to demonstrate that PKC in the myelin phosphorylates MBP, PKC was partially purified from rat CNS myelin by solubilization with Triton X-100 followed by a DEAE-cellulose column. MBP and histone III-S were phosphorylated in the presence of Ca²⁺ and phospholipid by rat myelin PKC. High voltage electrophoresis revealed that the phosphoamino acids in MBP by this kinase was serine residue, which is known to be the amino acid phosphorylated by PKC. The activity of PKC extracted from myelin was inhibited by the addition of psychosine to the incubation mixture. To confirm the presence of PKC molecule and to identify the isoform of PKC in the myelin, the solubilized myelin fraction was applied on SDS-PAGE, transferred to a nitrocellulose sheet and stained with anti-PKC monoclonal antibodies. Rat CNS myelin contained the PKC of about 80 kDa (intact PKC), and no proteolytic fragments were observed. PKC isozymes in myelin were type II and III. A developmental study from 14 to 42 postnatal days showed that PKC activity in CNS myelin seemed to parallel the deposition of myelin protein.     

Opioid receptor contributes to ischemic preconditioning through protein kinase C activation in rabbits

Recent studies have reported that protection from ischemic preconditioning (PC) is blocked by the opioid receptor antagonist naloxone (NAL). We tested whether an opioid agonist could mimic PC in the rabbit heart, whether that protection involved protein kinase C (PKC) activation, and whether opioid receptors act in concert with other PKC-coupled receptors. Rabbit hearts were subjected to 30min coronary occlusions and were reperfused for either 3 (in situ) or 2 (in vitro) h. Infarct size was determined by staining with triphenyltetrazolium chloride. In untreated in situ hearts 38.5 ± 1.6% of the risk zone infarcted. PC with 5 min ischemia/10 min reperfusion significantly limited infarction to 12.7 ± 2.9% (p < 0.01). NAL infusion did not modify infarction (39.6 ± 1.6%) in non-PC hearts, but blocked the effect of one cycle of PC (34.4 ± 3.6% infarction). NAL, however, could not block cardioprotection when PC was amplified with 3 cycles of ischemia/reperfusion (9.9 ± 1.4% infarction, p < 0.01 vs. control). Morphine could also mimic ischemic preconditioning, but only at a dose much higher than would be used clinically (3 mg/kg). In isolated hearts pretreatment with morphine (0.3 M) significantly limited infarction to 9.3 ± 1.2% (p < 0.01 vs. 32.0 ± 3.1% in controls). This cardioprotective effect of morphine could be blocked by either the PKC inhibitor chelerythrine (30.4 ± 2.6% infarction) or NAL (34.0 ± 2.6% infarction). Neither chelerythrine nor NAL by itself modified infarction in non-PC hearts. NAL could not block protection from one cycle of PC in isolated hearts indicating that an intact innervation may be required for endogenous opioid production. Thus, opioid receptors, like other PKC-coupled receptors, participate in the triggering PC in the rabbit heart.     

BiP,an endoplasmic reticulum chaperone,modulates the development of morphine antinociceptive tolerance

Morphine is a potent analgesic, but the molecular mechanism for tolerance formation after repeated use is not fully understood. Binding immunoglobulin protein (BiP) is an endoplasmic reticulum (ER) chaperone that is central to ER function. We examined knock‐in mice expressing a mutant BiP with the retrieval sequence deleted in order to elucidate physiological processes that are sensitive to BiP functions. We tested the thermal antinociceptive effect of morphine in heterozygous mutant BiP mice in a hot plate test. Paw withdrawal latencies before and after a single administration of morphine were not significantly different between the wild‐type and mutant BiP mice. Repeated morphine administration caused the development of morphine tolerance in the wild‐type mice. The activation of glycogen synthase kinase 3b (GSK‐3b) was associated with morphine tolerance, because an inhibitor of GSK‐3β prevented it. On the other hand, the mutant BiP mice showed less morphine tolerance, and the activation of GSK‐3b was suppressed in their brain. These results suggest that BiP may play an important role in the development of morphine tolerance. Furthermore, we found that a chemical chaperone which improves ER protein folding capacity also attenuated the development of morphine tolerance in wild‐type mice, suggesting a possible clinical application of chemical chaperones in preventing morphine tolerance.     

Molecular interaction in the mouse PAG between NMDA and opioid receptors in morphine-induced acute thermal nociception

Previous evidence demonstrates that low dose morphine systemic administration induces acute thermal hyperalgesia in normal mice through μOR stimulation of the inositol signaling pathway. We investigated the site of action of morphine and the mechanism of action of μOR activation by morphine to NMDA receptor as it relates to acute thermal hyperalgesia. Our experiments show that acute thermal hyperalgesia is blocked in periaqueductal gray with the μOR antagonist CTOP, the NMDA antagonist MK801 and the protein kinase C inhibitor chelerythrine. Therefore, a site of action of systemically administered morphine low dose on acute thermal hyperalgesic response appears to be located at the periaqueductal gray. At this supraspinal site, μOR stimulation by systemically morphine low dose administration leads to an increased phosphorylation of specific subunit of NMDA receptor. Our experiments show that the phosphorylation of subunit 1 of NMDA receptor parallels the acute thermal hyperalgesia suggesting a role for this subunit in morphine-induced hyperalgesia. Protein kinase C appears to be the key element that links μOR activation by morphine administration to mice with the recruitment of the NMDA/glutamatergic system involved in the thermal hyperalgesic response.     

Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1–7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of G_q/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer.     

蛋白激酶C抑制剂Staurosporine对HeLa细胞周期的影响

用蛋白激酶C的抑制剂Staurosporine(10nmol/L)处理HeLa细胞,明显抑制HeLa细胞的增殖。这种抑制作用不是由于引起细胞死亡,而是因为细胞被阻断在G2期。这种阻断作用伴随着HeLa细胞多倍体的形成,提示Staurosporine抑制了HeLa细胞蛋白激酶C活性后引起的细胞阻滞,对细胞核的周期运转没有影响。进一步的探讨发现这种抑制作用可能是通过干扰细胞骨架的正确分布形成的,表明蛋白激酶C对于HeLa细胞由G2到M期正确过渡起重要作用。     

Cyclic AMP-dependent protein kinase (PKA) phosphorylates Ser362 and 467 and protein kinase C phosphorylates Ser550 within the M3/M4 cytoplasmic domain of human nicotinic receptor alpha4 subunits

Studies have suggested that the expression, translocation, and function of alpha4beta2 nicotinic receptors may be modulated by alpha4 subunit phosphorylation, but little direct evidence exists to support this idea. The objective of these experiments was to identify specific serine/threonine residues on alpha4 subunits that are phosphorylated in vivo by cAMP-dependent protein kinase and protein kinase C (PKC). To accomplish this, DNAs coding for human alpha4 subunits containing alanines in place of serines/threonines predicted to represent phosphorylation sites were constructed, and transiently transfected with the DNA coding for wild-type beta2 subunits into SH-EP1 cells. Cells were pre-incubated with (32)Pi and incubated in the absence or presence of forskolin or phorbol 12,13-dibutyrate. Immunoprecipitated alpha4 subunits were subjected to immunoblot, autoradiographic and phosphoamino acid analyses, and two-dimensional phosphopeptide mapping. Results confirmed the presence of two alpha4 protein bands, a major band of 71/75 kDa and a minor band of 80/85 kDa. Phosphoamino acid analysis of the major band indicated that only serine residues were phosphorylated. Phosphopeptide maps demonstrated that Ser362 and 467 on the M3/M4 cytoplasmic domain of the alpha4 subunit represent major cAMP-dependent protein kinase phosphorylation sites, while Ser550 also contained within this major intracellular loop is a major site for protein kinase C phosphorylation.     

Molecular Cloning and Expression of Starfish Protein Kinase C Isoforms

To investigate the roles of protein kinase C (PKC) isoforms in Echinoderms, we cloned starfish cDNAs for novel, atypical, and conventional PKCs. They showed highest homology with PKCδ, ι, and α isoforms respectively. It was predicted from the whole genome sequence and by RT-PCR that sea urchin has only one isoform of each PKC subgroups. It is thus likely that these isoforms are the prototypes or ancestors of the PKC subgroups. The phylogenetic tree suggests that atypical PKC was first formed by evolution from the common prototype of AGC protein kinase family, and novel and conventional PKCs next. RT-PCR analysis indicated that novel and atypical PKC mRNAs are expressed ubiquitously in all tissues of adult starfish, whereas conventional PKC mRNA is expressed mainly in the ovary and oocytes, and only slightly in the tube foot and stomach. Upon heterologous expression, only atypical PKC was expressed in the functional form in insect cells.     

Phospholipase C-protein kinase C mediated phospholipase D activation pathway is involved in tamoxifen induced apoptosis

Tamoxifen (TAM) is the endocrine therapeutic agent the most widely used in the treatment of breast cancer, and it operates primarily through the induction of apoptosis. In this study, we attempted to elucidate the non-ER mediated mechanism behind TAM treatment, involving the phospholipase C-protein kinase C (PLC-PKC) mediated phospholipase D (PLD) activation pathway, using multimodality methods. In TAM treated MCF7 cells, the PLC and PLD protein and mRNA levels increased. Phosphatidylethanol (PEt) and diacylglycerol (DAG) generation also increased, showing increased activity of PLD and PLCgamma1. Translocation of PKCalpha, from cytosol to membrane, was observed in TAM treated cells. By showing that both PKC and PLC inhibitors could reduce the effects of TAM-induced PLD activation, we confirmed the role of PKC and PLC as upstream regulators of PLD. Finally, we demonstrated that TAM treatment reduced the viability of MCF7 cells and brought about rapid cell death. From these results, we confirmed the hypothesis that TAM induces apoptosis in breast cancer cells, and that the signal transduction pathway, involving PLD, PLC, and PKC, constitutes one of the possible mechanisms underlying the non-ER mediated effects associated with TAM.